Regulation of the pyrimidine biosynthetic pathway by lysine acetylation of E. coli OPRTase.

The de novo pyrimidine biosynthesis pathway is an important route due to the relevance of its products, its implications in health and its conservation among organisms. Here, we investigated the regulation by lysine acetylation of this pathway. To this aim, intracellular and extracellular metabolites of the route were quantified, revealing a possible blockage of the pathway by acetylation of the OPRTase enzyme (orotate phosphoribosyltransferase). Chemical acetylation of OPRTase by acetyl-P involved a decrease in enzymatic activity. To test the effect of acetylation in this enzyme, K26 and K103 residues were selected to generate site-specific acetylated proteins. Several differences were observed in kinetic parameters, emphasizing that the kcat of these mutants showed a strong decrease of 300 and 150-fold for OPRTase-103AcK and 19 and 6.3-fold for OPRTase-26AcK, for forward and reverse reactions. In vivo studies suggested acetylation of this enzyme by a nonenzymatic acetyl-P-dependent mechanism and a reversion of this process by the CobB deacetylase. A complementation assay of a deficient strain in the pyrE gene with OPRTase-26AcK and OPRTase-103AcK was performed, and curli formation, stoichiometric parameters and orotate excretion were measured. Complementation with acetylated enzymes entailed a profile very similar to that of the ∆pyrE strain, especially in the case of complementation with OPRTase-103AcK. These results suggest regulation of the de novo pyrimidine biosynthesis pathway by lysine acetylation of OPRTase in Escherichia coli. This finding is of great relevance due to the essential role of this route and the OPRTase enzyme as a target for antimicrobial, antiviral and cancer treatments.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

Primary Study of Producing UMP by Overexpressing URA 5 and URA 3 Genes in Saccharomyces cerevisiae / 中国生物工程杂志

To increase the biotransfomation efficiency from the orotic acid to the uridine 5'-monophosphate(UMP),URA5 gene encoding orotate phosphoribosytransferase was amplified from Saccharomyces cerevisiae BY4742 by PCR,then it was inserted into the expression vector pYX212(contained orotidine monophosphate decarboxylase gene URA3)and the pYX212-URA5 was transformed into Saccharomyces cerevisiae BJX12 by electroporation.The recombinant strain was elementarily used to convert orotic acid to UMP.The results showed that pYX212-URA5/BJX12 could accumulate 7mmol/L UMP from 32mmol/L orotic acid in 26h,significantly higher than both control groups pYX212/BJX12(2.7mmol/L) and BJX12(2.4 mmol/L).